Complex, membrane-spanning proteins are difficult to express in host cells. In general, these proteins are toxic to the host, and various expression systems yield low quantities of expressed protein. In addition, these membrane-spanning polypeptides are difficult to solubilize, with aggregation and denaturing causing difficulties in producing a protein product of sufficient quality and quantity for effective use.
One example of a membrane-spanning protein is the 4-domain subfamily A (MS4A) gene family that includes CD20, high affinity IgE receptor β chain, HTm4, and the like. These proteins are structurally related, at least in the four membrane-spanning domains in the cell surface (Ishibashi et al., 2001, Gene 264:87-93). Although overall amino acid sequence identity ranges from 25-40% in the polypeptides of the MS4A family, amino acids of the first three membrane-spanning domains share higher identity and homology than the overall polypeptide (Ishibashi et al., 2001, Supra; Liang et al., 2001, Genomics 72:119-127). Structurally, the MS4A polypeptides also share a common motif of an extracellular loop. Both the N- and C-termini of the MS4A polypeptide are found on the cytoplasm side of the cell membrane (Ishibashi et al., 2001, Supra). The N- and C-termini display a much greater sequence divergence among polypeptides of the MS4A gene family (Ishibashi et al., 2001, Supra).
Despite many structural similarities, polypeptides of the MS4A gene family are not uniformly expressed in individual cell types (Liang et al., 2001, Supra). CD20 is expressed exclusively in B cells (Stashenko et al., 1980, J. Immunol., 125: 1678-1685). High-affinity IgE receptor β chain (FcεRIβ) is expressed exclusively in mast cells and basophils (Kinet, 1999, Annu. Rev. Immunol., 17: 931-972). FcεRIβ binds IgE and mediates intracellular signaling (i.e., degranulation) triggered by antigen binding (Dombrowicz et al., 1998, Immunity, 8: 517-529; Lin et al., 1996, Cell, 85: 985-995). HTm4 is expressed in hematopoietic tissue and serves as a hematopoietic cell cycle regulator (Donato et al., 2002, J. Clin. Invest., 109: 51-58). These proteins do share a common feature, the complex structure of membrane-spanning peptides. This feature makes the protein very difficult to express in a host cell and to solubilize from the cell membrane in a “native” configuration.
Membrane-spanning polypeptides, for example CD20, are potential targets for therapeutics in the treatment of diseases such as cancer and autoimmune diseases. CD20 was first identified as a marker for B cells over 20 years ago and is now established as a marker present on the majority of B cell lymphomas. CD20 is a target for monoclonal antibody therapy in the treatment of non-Hodgkins lymphoma (NHL), and specifically, it is the target for the chimeric antibody rituximab (RITEUXAN®), a lead therapeutic in the treatment of NHL. Rituximab recognizes CD20 expressed on B cells. Binding of rituximab is conformation-dependent and hinds to CD20 having dependent, loop structure between the third and fourth transmembrane helical regions containing cysteine residues at positions 167 and 183.
A significant hurdle in the development of therapeutics that target membrane-spanning polypeptides, such as CD20, is the inability to produce sufficient quantities of these polypeptides in host cells, particularly in bacterial cells, and the inability to produce purified recombinant or naturally occurring membrane-spanning polypeptides in a native conformation. Methods for producing and solubilizing naturally occurring and/or recombinant membrane-spanning polypeptides in a native conformation are needed.